Inhibitory gamma-aminobutyric acid type A (GABAA) receptors in the central nervous system are major targets of the potent general anesthetics etomidate, propofol, barbiturates, and neuroactive steroids. These amphiphilic drugs stabilize conducting GABAA receptor conformations, enhancing inhibitory transmission, and reducing neuronal activity. Molecular level structural data about these drug-receptor interactions will help guide development of more selective, safer anesthetics. The overall aim of this proposal is to better define the interactions of propofol, etomidate, alphaxalone and pentobarbital at intramembrane subunit-subunit interfacial pockets on GABAA receptors. The data we propose to obtain will advance our understanding of how transmembrane domains at subunit interfaces rearrange during receptor channel activation, the number of anesthetic sites they harbor, and how they interact with different potent anesthetics. In addition, we anticipate identifying new GABAA receptor mutations for future experiments aimed at defining anesthetic mechanisms in transgenic animals. Our novel working hypothesis is that all five GABAA receptor subunit interfaces formed by transmembrane M1 and M3 domains on adjacent subunits form amphiphilic anesthetic-binding pockets that change shape during ion channel gating. Hetero-pentameric synaptic GABAA receptors containing 13, 21, and 22 subunits form four distinct types of interfacial pockets that may selectively interact with different anesthetics. Individual types of interfacial pockets also may contain multiple sub-sites for different anesthetics, some partially overlapping. We propose structural studies of the transmembrane interfaces between GABAAR 11, 22, and 32L subunits to test critical aspects of this hypothesis. These include: Aim 1) identifying residues in interfacial pockets that are accessible to small amphiphilic compounds, in open vs. closed receptors, and establishing the orientation of the M1 and M3 domains at these interfaces;Aim2) indentifying residues in these pockets that influence ion channel opening and closing in the absence and presence of GABA;and Aim 3) identifying residues in these pockets that influence sensitivity to the actions of potent general anesthetics, and testing for proximity between anesthetics and putative binding determinants. Our experimental strategy employs scanning cysteine and tryptophan mutants and electrophysiology in recombinant GABAA receptors in two expression systems, and exploits sulfhydryl-specific chemistry in cysteine mutants. Electrophysiological data will be analyzed using global functional allosteric models and further interpreted in the context of evolving homology models of GABAAR structure. Structural models will be refined using cysteine accessibility and both protection results and models will be used to generate new testable hypotheses. PUBLIC HEALTH RELEVANCE: General anesthetics are among the most beneficial and most dangerous drugs in routine clinical use, yet the mechanisms of their actions remain poorly understood. Detailed mechanistic studies are needed to guide development of improved drugs that could substantially reduce both risks and costs of anesthesia care for many millions of patients each year. The proposed experiments will advance our understanding of where and how potent general anesthetics (etomidate, propofol, pentobarbital, and alphaxalone) act on their major molecular targets in the nervous system, gamma-aminobutyric acid type A receptors, by testing the novel hypothesis that molecular binding sites for these drugs are formed at multiple subunit-subunit interfaces within neuronal cell membranes (intra-membrane subunit interfaces).